A randomized, placebo-controlled clinical trial evaluating of a mouthwash containing Sambucus williamsii var. coreana extract for prevention of gingivitits

The purpose of this study is to verify the clinical applicability by applying a mouthwash containing Sambucus williamsii var. coreana extract for preventing periodontal disease. A randomized, double-blind, placebo-controlled study was conducted on 64 patients, excluding those with insufficient data, who visited M dental clinic located in Busan, Korea. Thirty-two people were assigned respectively to the saline solution gargle group and the Sambucus williamsii var. coreana extract gargle group to conduct the O'Leary index, plaque index (PI), gingival index (GI), and subgingival plaques. For the homogeneity of the two groups, scaling was carried out one week before the experiment, and the participants were taught for oral care to conduct during the study period. SPSS 24.0 for Windows (IBM Corp., Armonk, NY, USA) was used to compare the saline solution gargle group and the Sambucus williamsii var. coreana extract gargle group as well as to analyze Baseline (before gargle application), Treatment (immediately after gargle application), and After 5 Days (5 days after gargle application). There was a significant difference in the O'Leary index, PI, GI and subgingival plaques after Treatment and After 5 days (p < 0.05). Also, the periodontal-related indexes improved as the application time increased in the Sambucus williamsii var. coreana extract gargle group. The antibacterial effect was also shown for gram-positive bacteria and gram-negative bacteria in subgingival plaques as the application time increased. The use of the mouthwash containing Sambucus williamsii var. coreana extract was found to be effective for oral periodontal-related indicators and bacteria causing periodontal disease. Therefore, using a mouthwash containing Sambucus williamsii var. coreana extract, a natural drug, will possibly maintain healthy periodontal health by inhibiting and preventing the progression of periodontal disease.

Study participants. The G* Power 3.1 program calculated sample sizes. 68 participants were required for an independent t-test with significance level a = 0.05 two-tailed, power = 0.8, and effect size = 0.7. An initially planned sample size was 96 to consider a dropout rate of 40%, and actual participants were 98 in this study. The dropout rate was set high as the subjects were college students or working adults. 98 subjects were screened, but 74 participants were randomly assigned to the saline group or Sambucus williamsii var. coreana extract group after excluding 24 subjects who did not meet inclusion criteria or refused to participate during 5 days. 64 subjects were selected as final subjects, excluding 6 subjects who did not follow the guidelines for 5 days and 4 subjects who did not complete the final analysis (Fig. 1). www.nature.com/scientificreports/ Study design and protocol. An experiment was randomized, double-blind, and placebo-controlled. In this study, a dental hygienist with more than ten years of experience directly stated the aim of the study to the patients who visited M dental clinic in Busan from October 2020 to June 2021. These patients agreed to participate in the study on December 15, 2020. Among the subjects, the following subjects who met the exclusion criteria were excluded from this study: a person with severe dental disease (e.g., periodontitis, dry mouth, dental caries), a person with a disease that may cause bad breath (e.g., Sjogren's syndrome, rheumatism, renal disease, and hepatic disease), a smoking person, a person diagnosed with sinusitis or rhinitis, a person taking antibiotics, a person with tongue problems (e.g., tongue cancer, glossitis), a person received scaling within 2 months. Among dental caries, patients with enamel caries were eligible to participate in the study, but patients with more than one dentin caries were excluded. Accordingly, the subjects of this study were those who agreed to complete the questionnaire, were not included in the exclusion criteria, and had 16 or more remaining teeth. 74 patients who satisfied the inclusion criteria were selected as the study subjects. Finally, the participants in this study were analyzed with a result of 64 subjects.

Clinical examination.
Participants who agreed to this study visited the M dental clinic in Busan one week before the start and received an oral examination by a dentist. Two dental hygienists trained under the guidance of a dentist performed light scaling on the participants for the same oral environment and provided the same toothbrush and toothpaste to them. After a recovery period of 1 week after scaling, the study started. As representative teeth for an oral examination, maxillary right first molar (#16), maxillary left central incisor (#21), maxillary left first premolar (#24), mandibular left first molar (# 36), mandibular right central incisor (#41), and mandibular right first premolar (#44) were selected and measured. After scaling, two dental hygienists trained under the guidance of a dentist obtained the following data: before the application of mouthwash as Baseline, immediately after the application of mouthwash as Treatment, and five days after the application of mouthwash as After 5 Days. Participants were provided with labeled items so that they could not tell whether they were in the experimental group or the control group. They were also educated on how to use mouthwash and how often and how to brush their teeth using the provided toothpaste and toothbrush. For five days, the experimental group held 15 ml of mouthwash containing Sambucus williamsii var. coreana extract for 30 s and then spat it out before going to bed after brushing, while the control group did the same with 15 ml of saline. After gargling, intake of food, including water, was prohibited. O'Leary index, plaque index (PI), gingival index (GI), and microbiological analysis were evaluated as indicators related to periodontal disease.   24 , where all teeth in the oral cavity were discolored with a dental surface discoloration agent and estimated the status of adherence (%) using the plaque control score (O'Leary index). Score 1 if the dental plaque adheres to the dental surfaces of 4 teeth (mesial, efferent, facies, and lingual), and score 0 if not.

PI.
Using the PI technique of Loe and Silness 25 , dividing all teeth surface into two parts; the gingival margin and the tooth surface. Then, measuring plaque accumulation and its thickness with a red colorant. The evaluation criteria are as follows; 0 = no plaque; 1 = thin plaque attached to the gingival margin and visible when rubbing gently with a probe or applying a tooth colorant; 2 = moderate plaque visible along the gingival margin; 3 = thick plaque collection in gingival margin and tooth surface as well as in the gingival pockets. PI is the average quantity of plaque per measured tooth surface, and its score for each tooth was calculated using the average value.
GI. Gingival health status at the proximal, distal, buccal, and lingual regions of all teeth was evaluated using the GI technique 26 . Each section was assigned from score 0 to 3: 0 = normal gingiva; 1 = a slight color change and swelling of gingivitis showing no bleeding with a mild stimulus; 2 = red and swollen gingivitis showing bleeding with a mild stimulus; 3 = remarkably red and swollen gingivitis due to progressed inflammation and showing the possibility of ulceration and natural bleeding. The values for each tooth were added up to calculate the individual's total mean GI.
Microbiological analysis. To obtain subgingival microbiome samples from periodontal pockets, sterile #15 paper points were placed in the gingival sulcus at four sites: two maxillary teeth (#16 and #21) and two mandibular teeth (#36 and #41) with pocket depth (PD) less than 4 mm for 10 s. They were then placed in sterile 1.5 mL tubes and frozen at − 20 °C until right before an analysis. DNA was dragged from collected #15 paper points using the AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, Korea) following the manufacturer's instructions. As shown in Supplementary

Results
Population characteristics. Clinical indicators related to periodontal disease. www.nature.com/scientificreports/ Distribution of periodontal disease-causing bacteria in the study subjects. Table 3 shows the results of confirming the distribution of bacteria in the oral cavity of the study subjects. Among Gram-positive bacteria, those with P. micra had the highest proportion in the oral, and the average of bacteria was also high. Among Gram-negative bacteria, those with F. nocleatum had the highest proportion in the oral, and the average of bacteria was also high. www.nature.com/scientificreports/ Gram-positive bacteria in subgingival plaques. Gram-positive bacterial data in subgingival plaques was detected as three types of bacteria: P. micra, Staphylococcus aureus (S. aureus), and Eubacterium nodatum (E. nodatum). In addition, the effect difference between the two groups was verified according to the maxillary and mandible (Table 4). When comparing the saline gargle group and the Sambucus williamsii var. coreana extract gargle group against P. micra, there was a significant difference only in the mandible at the time of 'Treatment' and ' After 5 days' (p < 0.05). There was a significant difference in ' After 5 days' in the maxilla (p < 0.05) regarding S. aureus; it was not detected in both groups in the mandible. There was no significant difference between the two groups for E. nodatum in the oral cavity (p > 0.05). Also, based on the 'Baseline' , there was a significant difference from 'Treatment' to ' After 5 days' in the mandible of the Sambucus williamsii coreana var. extract gargle group in the case of P. micra (p < 0.05). As for E. nodatum, there was a significant difference in both groups in the maxilla (p < 0.05).
Gram-negative bacteria in subgingival plaques. The following seven types of bacteria were detected as gram-negative bacteria in the oral cavity: P. gingivalis, T. denticola, F. nocleatum, P. intermedia, P. nigrescens, Eikenella corrodens (E. corrodens), and Campylobacter rectus (C. rectus). In addition, the effect difference between the two groups was verified according to the maxillary and mandible (Table 5). When comparing the saline gargle group and the Sambucus williamsii var. coreana extract gargle group, P. gingivalis showed a significant difference at 'Treatment' and ' After 5 days' in maxilla and mandible (p < 0.05). T. denticola was detected only in the mandible, showing a significant difference between the two groups at 'Treatment' and ' After 5 days' (p < 0.05). F. nocleatum differed between groups only in the maxilla ' After 5 days' (p < 0.05). As for P. intermedia showed a significant difference between the groups at 'Treatment' and ' After 5 days' only in the mandible (p < 0.05). In the case of P. nigrescens, there was a significant difference between the two groups ' After 5 days' in both maxilla and mandible (p < 0.05). E. corrodens was not significantly different between the two groups in the maxilla and mandible. C. rectus showed a difference between the two groups at 'Treatment' and ' After 5 days' in the case of maxilla but showed a significant difference only at 'Treatment' in the mandible (p < 0.05). According to the application time, the changes were not significantly different in the saline gargle group from 'Baseline' to ' After 5 days' except for F. nocleatum in the maxilla and E. corrodens in the mandible, among the seven types of gram-negative bacteria (p > 0.05). On the other hand, in the Sambucus williamsii var. coreana extract gargle group, all bacteria in the maxilla and mandible were significantly reduced according to the application time, www.nature.com/scientificreports/ except for F. nocleatum in the mandible; P. intermedia and P. nigrescens of the maxilla. In particular, based on 'Baseline' , P. gingivalis and C. rectus showed a marked differences from 'Treatment' to ' After 5 days' in the maxilla and mandible, verifying that gram-negative bacteria decreased clearly. From 'Treatment' to ' After 5 days' , T. denticola, P. intermedia, and E. corrodens were reduced in the mandible only, and T. denticola was not even detected at 'Treatment' and ' After 5 days' .

Discussion
About 700 types of microorganisms live in the oral cavity; because the nutrients necessary for the growth of microorganisms are continuous, the bacteria proliferate to form dental plaques that cause oral bacterial diseases 28 . Therefore, the best way to prevent this is to remove the dental plaque, representatively using Tetracyclines, Metrodinidazole, Penicillin, Clindamycin, and Ciprofloxacin out of the various types of antibacterial agents 29 . These chemical antibacterial agents inhibit bone loss and are effective for periodontitis 30 , but their use is limited due to side effects such as the generation of resistant bacteria and mycosis 31 . Therefore, this study verified the O'Leary index, PI, and GI as periodontal-related indicators; and antibacterial effects on pathogens causing periodontal diseases to develop a natural mouthwash using Sambucus williamsii var. coreana extract, which has a natural antibiotic effect.
The high baseline values of the PI, GI, and bacteria seem to come out due to the lack of oral care before applying mouthwash. After measuring the baselines, two examiners (dental hygienists) also educated the subjects about how to use mouthwash and how often and how to brush teeth using the provided toothpaste and toothbrush. As a result of this study, O'Leary index, PI, and GI all showed marked decreases from 'Treatment' to ' After 5 days' compared to the saline gargle group when applying mouthwash containing Sambucus williamsii var. coreana extract. Also, as the application time of the mouthwash containing Sambucus williamsii var. coreana extract increased, the values of periodontal-related indicators were effectively lowered, verifying that there was an effect of improving the oral environment.
The immediate improvement in GI in this study is shown as a statistical result by a small number of subjects with a large number of subjects with healthy gingiva and a large change in GI improvement.
According to Cai et al. 's study 32 , herb mouthwash has mentioned potential benefits in plaque and inflammation control as a supplement to routine oral hygiene in patients with gingivitis, and in particular, Balappanavar et al. 's study 33 and Meena Priya et al. 's study 34 showed a greater reduction in gingivitis after intervention.
According to a previous study, Sambucus williamsii var. coreana extract had antioxidant and anti-inflammatory effects and had no cytotoxicity 35 . Therefore, applying it in the oral cavity as a mouthwash is considered appropriate to suppress and prevent periodontal disease. P. micra, which is known as a bacterium associated with periodontal disease, is a gram-positive, non-motile, anaerobic coccus 36 that is more common in patients with severe or moderate periodontitis than those with gingivitis and mild periodontitis 37 . In this study, a mouthwash containing Sambucus williamsii var. coreana extract was effective in the mandible; the antibacterial effect appeared immediately after application and continued until ' After 5 days' . P. micra is known to act as a core pathogen in the development of the periodontal disease 38 ; signaling molecules derived from P. micra stimulate the expression of the P. gingivalis proteolytic gingipain enzyme thirteen times more to enhance the growth and toxicity of Porphyromonas gingivalis, a major periodontal pathogen 39 . P. gingivalis, a gram-negative anaerobic bacterium, is associated with periodontal destruction and increasing risk of disease recurrence 40 ; it is the foremost periopathogen for periodontal disease progression 41 . After applying the mouthwash containing Sambucus williamsii var. coreana extract to P. gingivalis, it decreased sharply in the maxilla and the mandible compared to the saline gargle group. The mouthwash containing Sambucus williamsii var. coreana extract was verified to be very effective against P. gingivalis as it decreased continuously right after applying the mouthwash. Therefore, a mouthwash using Sambucus williamsii var. coreana extract can inhibit and block the growth of core pathogens of periodontal disease. P. gingivalis is also considered the major pathogen because of its ability to change the normal microflora into a dysbiotic community 42 . Among dysbiotic species, the increase in P. gingivalis, a major periodontal disease-related pathogen, is accompanied by various bacteria, including P. intermedia and F. nucleatum 43 . Since the antibacterial effect was shown by a marked reduction of P. gingivalis in this study, mouthwash containing Sambucus williamsii var. coreana extract will play a huge role in reducing various anaerobic bacteria. P. intermedia, the anaerobic black bacteria, is one of the powerful causative agents of adult-type periodontitis and various types of periodontal disease 44 . In addition, since the expression frequency of P. nigrenscens was reported to be more than double that of P. intermedia in the inflammatory site of patients with periodontal disease 45 , dealing with P. nigrenscens is also necessary. Compared with the saline Table 5. Gram-negative bacterial measurements in subgingival plaque (7 types). ￥ p-values are determined by independent t-test, *p-values are determined by one-way ANOVA and different letters (a and b) by the presented statistically significant result of the post hoc Tukey HSD (P < 0.05). Values are means ± standard deviations. Significant (bold).

Variables Group
Mean ± SD www.nature.com/scientificreports/ gargle group, P. intermedia effectively decreased in the mandible immediately after applying a mouthwash containing Sambucus williamsii var. coreana extract. There was no effect on P. nigrenscens immediately after using a mouthwash containing Sambucus williamsii var. coreana extract, but it became effective in both the maxilla and mandible after using the mouthwash for five days. Both P. intermedia and P. nigrenscens more effectively decreased over time in the mandible as applying a mouthwash containing Sambucus williamsii var. coreana extract. As a result, a mouthwash containing Sambucus williamsii var. coreana extract reduced P. gingivalis immediately after applying a mouthwash. P. nigrenscens and P. intermedia also decreased as the application time increased. F. nocleatum, one of the oral microflora and a causative agent of periodontitis, is also related to preterm birth, colorectal cancer, inflammatory bowel disease, respiratory tract infections, cardiovascular disease, rheumatoid arthritis, and Alzheimer's disease 46 . T. denticola, a bacterium that causes progressive periodontitis, is associated with losing attached gingiva and bleeding when measuring the periodontal pocket and its depth 3 48 , so continuous management of anaerobic bacteria related to periodontal disease is required. Based on the study results, a mouthwash containing Sambucus williamsii var. coreana extract reduced bacteria in the oral cavity such as P. gingivalis, C. rectus, T. denticola, P. intermedia, and E. corrodens immediately after application. As the application time of a mouthwash containing Sambucus williamsii var. coreana extract extended, a more marked antibacterial effect appeared, which means it is excellent in improving periodontal disease. According to the results of this study, mouthwash was more effective in the mandible; it seems that the teeth and periodontal tissues of the mandible were in contact with and stored more in the mouthwash when keeping the mouthwash in the mouth. Therefore, to maximize the effect of mouthwash, it will be more effective to gargle mouthwash enough to cover the entire oral cavity rather than simply holding it.
The limitation of this study is that a mouthwash containing Sambucus willamsii var. coreana extractwas applied only for five days to verify its effects. For this reason, it can be considered that the use of mouthwash for a short period of time alone has the effect of improving gingivitis. However, further research is needed through clinical efficacy verification to confirm the improvement of periodontitis by expanding the subjects to periodontal disease patients and lengthening the period of mouthwash use. In addition, more systemic disease variables will be corrected in order to secure the homogeneity of study subjects for future research, and the reliability of the results of clinical trials will be secured through 1:1 propensity score matching.
Based on this study verifying the practicality and development of oral health care products using Sambucus williamsii var. coreana extract, a mouthwash containing Sambucus williamsii var. coreana extract can inhibit and prevent periodontal disease. As a natural ingredient with sufficiently excellent effects, Sambucus williamsii var. coreana extract can be used for oral health by improving periodontal disease.

Conclusion
A mouthwash containing Sambucus williamsii var. coreana extract affects oral hygiene by reducing the O'Leary index, PI, and GI. It also has a potential advantage in inflammation control by reducing periodontal diseasecausing bacteria, so alternative oral care products as the adjuvant treatment of periodontal disease can be possible. In addition, medicine using Sambucus williamsii var. coreana extract rather than chemicals can be a promising field for treating gingivitis.

Data availability
The data sets generated and/or analyzed during the current study are not publicly available for reasons of personal and organizational integrity but are available from the corresponding author on reasonable request. www.nature.com/scientificreports/